As described in the U.S. Patent to Burr et al, U.S. Pat. No. 6,079,836 (hereinafter referred to as the '836 patent), entitled: “Flow Cytometer Droplet Break-Off Location Adjustment Mechanism,” assigned to the assignee of the present application and the disclosure of which is incorporated herein, flow cytometers, such as that shown diagrammatically in FIG. 1, are commonly employed in the medical industry to analyze particles in a patient's body fluid (e.g., blood cells) as an adjunct to the diagnosis and treatment of disease. As a non-limiting example, in the course of chemotherapy treatment, such instruments may be used to sort and collect healthy blood cells (stem cells) from a quantity of blood that has been removed from the patient's bone marrow prior to chemotherapy. Once a chemotherapy treatment session has been completed, a collected quantity of these cells is then reinjected back into the patient, to facilitate migration and healthy blood cell regeneration.
For this purpose, in the flow cytometer system shown in FIG. 1, particles to be analyzed, such as cells of a centrifuged blood sample stored in a container 11, are injected into a (pressurized) continuous or uninterrupted stream of carrier fluid (e.g., saline) 12. The carrier fluid stream is directed along a flow channel 13 of a fluid flow chamber 14. The fluid flow channel 13 is intersected at an ‘upstream’ location 15 by an output light beam 16 emitted by an optical illumination subsystem, such as a one or more lasers 17. Located optically in the path of the laser output beam 16 after its being intercepted by the carrier fluid stream are one or more photodetectors of a photodetector subsystem 20. The photodetector subsystem is positioned to receive light that has been modulated by the contents of (particles/cells within) the fluid stream, including light reflected off a cell, the blocking of light by a cell, and a light emission from a fluorescent dye antibody attached to a cell.
In order to avoid confusion as to which photodetector output signal is representative of which illuminated cell, the fluid flow chamber 13 through the cytometer flow chamber is configured and sized to pass the particles or cells only one cell at the time through the intersection location 15 with the laser's output beam 16. As a consequence, as output signals from the photodetector subsystem 20 are modulated by particles transported by the carrier fluid stream, each modulation signal can be associated with an individual cell. If the output of the photodetector subsystem 20 satisfies prescribed ‘sort’ criteria associated with one or more parameters of a desired cell, it is used to control the sorting of a droplet 23 of carrier fluid containing that cell by an electrostatic droplet sorter located ‘downstream’ of an exit port 18 of the fluid flow chamber.
The carrier fluid stream is converted into individual droplets by an acoustically (e.g., piezoelectric transducer) driven droplet generator 27, which is coupled to the fluid flow chamber. The fluid stream leaving the exit port 18 proceed as an interconnected droplet stream 22 and then break off into separate droplets at a location 25 downstream of the chamber exit port. Also there is a ‘sort’ delay between the time that a cell passes through the laser intersection location 15 and a subsequent time at which the last attached portion of the carrier fluid stream containing that particular cell actually physically separates or breaks off from the carrier fluid stream as a distinct droplet 23 traveling along a travel path 26.
The location 25 at which the droplets form downstream of the flow chamber exit port 18 may be adjusted by varying parameters of the droplet generator drive signal. The rate at which droplets are formed is governed by the frequency of the acoustic drive signal, and the droplets become synchronized with the frequency of the piezo vibration of the droplet generator 27. As a non-limiting example, the acoustic drive frequency applied to the droplet generator 27 may be on the order of from four to one hundred KHz, at a fluid pressure on the order of from three to seventy psi.
The photodetector output is typically digitized and then analyzed by a cell type mapping or identification algorithm executed by an associated supervisory control processor of the flow cytometer's control workstation 50. Based upon this analysis, the control processor supplies control signals to a charging and deflection control circuit 52 of the droplet sorter 24 to sort or abort the droplet. In order to controllably sort an individual droplet 23 that breaks off or separates from the fluid stream exiting the flow chamber's exit port 18, the droplet sorter employs an electrostatic charging collar 31, which surrounds the travel path 26 of the droplet sequence. The charging collar may comprise a metallic cylinder that is located so as to surround the location along the droplet sequence travel path 26 where the individual droplets 23 separate from the fluid stream, and is typically several droplets in (axial) length. The charging collar 31 is positioned vertically downstream of the fluid chamber exit port 18 and upstream of an associated set of electrostatic (opposite polarity, high voltage) deflection plates 33 and 35 between which the stream of charged droplets 23c pass as they travel downwardly and are either sorted along one or more sorting paths 36 into associated sorted droplet collection containers 41, or are allowed to pass unsorted along travel path into an aborted or discarded wasted container 43. The center position is not always a waste container—as it may alternatively be employed as a collection container for collecting the sample that went through the instrument, not to waste it.
Under the control of the cell analysis and sorting routine executed by the cytometer workstation 50, a prescribed charging voltage pulse of a given duration is selectively applied to the charging collar, so as to charge a droplet that should contain the cell to be sorted. As the selectively charged droplet passes between the two opposite polarity high voltage deflection plates 33 and 35, it is attracted to the plate with the opposite charge, while being simultaneously repelled by the plate with the same or like charge. This electrostatic steering action directs the charged droplet along a deflected travel path that is off axis to the unsorted droplet travel path and into a sorted droplet collection container 41.
As noted above, for any given cell or particle of interest within the fluid stream, there is a ‘sort’ delay between the time at which the photodetector subsystem 20 generates an output signal for a particular cell, and the time of the sorting pulse at which a droplet 23 containing the cell actually individually separates or breaks of from the fluid stream. Knowing the exact duration of this sort delay is critical to accurate sorting of the droplets, since only the last attached droplet that breaks off from the fluid stream at the time of the applied sort charging pulse will be deflected by the deflection plates and subsequently collected into the sorted droplet collection container.
In accordance with the invention described in the '836 patent the flow cytometer is provided with a feedback-based signal processing mechanism that is operative to maintain the droplet break-off point at an initially calibrated spatial location (within the droplet charged collar of the droplet sorting mechanism) by means of a downstream optical detector subsystem, that looks for gaps in the fluid droplet stream that have been created by the deflection of charged droplets. The difference between the times at which these gaps are detected at a prescribed downstream location in the path of the droplet stream and the times at which deflected droplets that created the gaps were charged at the droplet charge collar is compared with a calibration reference interval. Any difference between the two is employed to adjust the amplitude of the piezo drive to the droplet generator, as necessary, to bring the instrument back into calibration. It has also been determined that controlling the temperature of the fluid stream and the pressure of the fluid stream can be as or more effective in correcting any differences.